Three distinct 3-methylcytidine (m³C) methyltransferases modify tRNA and mRNA in mice and humans

Chemical RNA modifications are central features of epitranscriptomics, highlighted by the discovery of modified ribonucleosides in mRNA and exemplified by the critical roles of RNA modifications in normal physiology and disease. Despite a resurgent interest in these modifications, the biochemistry of 3-methylcytidine (m³C) formation in mammalian RNAs is still poorly understood. However, the recent discovery of trm141 as the second gene responsible for m³C presence in RNA in fission yeast raises the possibility that multiple enzymes are involved in m³C formation in mammals as well. Here, we report the discovery and characterization of three distinct m³C-contributing enzymes in mice and humans. We found that methyltransferase-like (METTL) 2 and 6 contribute m³C in specific tRNAs and that METTL8 only contributes m³C to mRNA. MS analysis revealed that there is an ∼30-40% and ∼10-15% reduction, respectively, in METTL2 and -6 null-mutant cells, of m³C in total tRNA, and primer extension analysis located METTL2-modified m³C at position 32 of tRNA^Thr isoacceptors and tRNA^Arg(CCU) We also noted that METTL6 interacts with seryl-tRNA synthetase in an RNA-dependent manner, suggesting a role for METTL6 in modifying serine tRNA isoacceptors. METTL8, however, modified only mRNA, as determined by biochemical and genetic analyses in Mettl8 null-mutant mice and two human METTL8 mutant cell lines. Our findings provide the first evidence of the existence of m³C modification in mRNA, and the discovery of METTL8 as an mRNA m³C writer enzyme opens the door to future studies of other m³C epitranscriptomic reader and eraser functions.